f7: An α-helical peptide of p62 interrupts the TRB3/p62 interaction and promotes autophagy and UPS degradation.(a) HepG2 cells were treated with Pep2–A2 or Pep2-con (5 μM) for 12 h, and extracts were IP with anti-p62 Ab or normal rabbit IgG and blotted with anti-TRB3 Ab. (b,c) HEK 293T cells were transfected with the indicated plasmids. The effect of Pep2–A2 on the LC3/p62 and Ub/p62 binding was evaluated with Co-IP assay. (d) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h, and the colocalization of the P62/TRB3 was examined by immunostaining. Scale bar, 10 μm. (e) HepG2 cells were treated with Pep2-con, Pep2–A2 or Pep2–A2mut (5 μM) for 12 h and cell extracts were IP with anti-p62 Ab and blotted with anti-TRB3 Ab. (f) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h and the autophagy-associated proteins were detected by immunoblotting. (g) HepG2 cells infected with mRFP-GFP-LC3 plasmid were treated with Pep2–A2 or Pep2-con for 24 h, and the images were captured with confocal microscopy. Scale bar, 10 μm. See also Supplementary Movies 4–6 for details. (h) HepG2 cells transfected with UbG76V-GFP plasmid were treated with Pep2–A2 or Pep2-con for 24 h. Cell lysates were collected for immunoblotting analysis. (i) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h. The expressions of indicated proteins were detected by immunoblotting. (j) HepG2 cells were treated with Pep2-con, Pep2–A2, Pep2–A2 plus bafilomycin (left) or Pep2–A2 plus MG132 (right) for 12 h. The expression of indicated proteins was detected by immunoblotting. For all panels, n=3 independent experiments. Data indicate mean±s.e.m. Statistical significance was determined with one-way ANOVA; **P<0.01.

Mentions:
To verify whether A2 interrupts the TRB3/p62 interaction in tumour cells, a fused peptide Pep2–A2 was designed by linking a cell-penetrating peptide34 to A2 using a glycine–glycine linker. Treatment of HepG2 cells with Pep2–A2 inhibited the TRB3/p62 interaction (Fig. 7a) and rescued the TRB3-reduced association of P62 with LC3 and ubiquitinated proteins (Fig. 7b,c). Thus, Pep2–A2 inhibited the colocalization of TRB3 and p62 with the reduction of TRB3 and p62 (Fig. 7d). Moreover, Pep2–A2mut peptide carrying three, four, seven and nine alanine substitutions lost the binding capability with TRB3 (Fig. 6f) and showed less interfering effect on the p62/TRB3 interaction (Fig. 7e). It had reported that interactions of p62 with NBR1, RIP1, TRAF6 and Keap1 can regulate a diversity of cellular activities (Supplementary Fig. 6a)2435. We found that Pep2–A2 did not interrupt the interactions of p62 with NBR1 or RIP1 but enhanced the interactions of p62/TRAF6 and p62/Keap1 in HepG2 cells (Supplementary Fig. 6b–e). In addition, Pep2–A2 did not affect the interaction of TRB3 with AKT and ATF4, two reported binding partners of TRB3 (Supplementary Fig. 6f). Interestingly, treatment of cancer cells with Pep2–A2 activated autophagic signalling and flux (Fig. 7f,g and Supplementary Movies 7–9), suggesting that Pep2–A2 is an autophagy-inducing peptide through interrupting the TRB3/P62 interaction. Moreover, Pep2–A2 activated UPS in these cells (Fig. 7h). We therefore explored whether interrupting this interaction produced anticancer actions. We found that Pep2–A2 decreased the expression of several tumour-promoting factors (Fig. 7i), which could be reversed by bafilomycin or MG132 (Fig. 7j), suggesting that Pep2–A2 reduces expression of the tumour-promoting factors by activating both autophagy and UPS. In addition, Pep2–A2 inhibited proliferation, migration and invasion in basal and IGF-1-stimulated HepG2 cells (Fig. 8a–d). The similar antitumour roles were observed in the Pep2–A2-treated A549 and HCT-8 cells (Supplementary Fig. 6g). To further confirm whether it is the p62/TRB3 interaction responsible for tumour-promoting effects of TRB3, we constructed a p62mut expression vector that is p62-siRNA-resistant. This mutant lacks the C-terminal LIR and UBA domains and cannot interact with TRB3, but retains the self-oligomerization action. We found that the Pep2–A2 peptide showed no inhibiting effects on the proliferation and invasion of HepG2 cells with endogenous p62 depletion and isotopic expression of p62mut (Fig. 8e,f). Moreover, Pep2–A2mut peptide could not suppress tumour growth and invasion (Fig. 8g,h). These data demonstrate that the p62/TRB3 interaction is responsible for the tumour-promoting effects of TRB3.

f7: An α-helical peptide of p62 interrupts the TRB3/p62 interaction and promotes autophagy and UPS degradation.(a) HepG2 cells were treated with Pep2–A2 or Pep2-con (5 μM) for 12 h, and extracts were IP with anti-p62 Ab or normal rabbit IgG and blotted with anti-TRB3 Ab. (b,c) HEK 293T cells were transfected with the indicated plasmids. The effect of Pep2–A2 on the LC3/p62 and Ub/p62 binding was evaluated with Co-IP assay. (d) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h, and the colocalization of the P62/TRB3 was examined by immunostaining. Scale bar, 10 μm. (e) HepG2 cells were treated with Pep2-con, Pep2–A2 or Pep2–A2mut (5 μM) for 12 h and cell extracts were IP with anti-p62 Ab and blotted with anti-TRB3 Ab. (f) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h and the autophagy-associated proteins were detected by immunoblotting. (g) HepG2 cells infected with mRFP-GFP-LC3 plasmid were treated with Pep2–A2 or Pep2-con for 24 h, and the images were captured with confocal microscopy. Scale bar, 10 μm. See also Supplementary Movies 4–6 for details. (h) HepG2 cells transfected with UbG76V-GFP plasmid were treated with Pep2–A2 or Pep2-con for 24 h. Cell lysates were collected for immunoblotting analysis. (i) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h. The expressions of indicated proteins were detected by immunoblotting. (j) HepG2 cells were treated with Pep2-con, Pep2–A2, Pep2–A2 plus bafilomycin (left) or Pep2–A2 plus MG132 (right) for 12 h. The expression of indicated proteins was detected by immunoblotting. For all panels, n=3 independent experiments. Data indicate mean±s.e.m. Statistical significance was determined with one-way ANOVA; **P<0.01.

Mentions:
To verify whether A2 interrupts the TRB3/p62 interaction in tumour cells, a fused peptide Pep2–A2 was designed by linking a cell-penetrating peptide34 to A2 using a glycine–glycine linker. Treatment of HepG2 cells with Pep2–A2 inhibited the TRB3/p62 interaction (Fig. 7a) and rescued the TRB3-reduced association of P62 with LC3 and ubiquitinated proteins (Fig. 7b,c). Thus, Pep2–A2 inhibited the colocalization of TRB3 and p62 with the reduction of TRB3 and p62 (Fig. 7d). Moreover, Pep2–A2mut peptide carrying three, four, seven and nine alanine substitutions lost the binding capability with TRB3 (Fig. 6f) and showed less interfering effect on the p62/TRB3 interaction (Fig. 7e). It had reported that interactions of p62 with NBR1, RIP1, TRAF6 and Keap1 can regulate a diversity of cellular activities (Supplementary Fig. 6a)2435. We found that Pep2–A2 did not interrupt the interactions of p62 with NBR1 or RIP1 but enhanced the interactions of p62/TRAF6 and p62/Keap1 in HepG2 cells (Supplementary Fig. 6b–e). In addition, Pep2–A2 did not affect the interaction of TRB3 with AKT and ATF4, two reported binding partners of TRB3 (Supplementary Fig. 6f). Interestingly, treatment of cancer cells with Pep2–A2 activated autophagic signalling and flux (Fig. 7f,g and Supplementary Movies 7–9), suggesting that Pep2–A2 is an autophagy-inducing peptide through interrupting the TRB3/P62 interaction. Moreover, Pep2–A2 activated UPS in these cells (Fig. 7h). We therefore explored whether interrupting this interaction produced anticancer actions. We found that Pep2–A2 decreased the expression of several tumour-promoting factors (Fig. 7i), which could be reversed by bafilomycin or MG132 (Fig. 7j), suggesting that Pep2–A2 reduces expression of the tumour-promoting factors by activating both autophagy and UPS. In addition, Pep2–A2 inhibited proliferation, migration and invasion in basal and IGF-1-stimulated HepG2 cells (Fig. 8a–d). The similar antitumour roles were observed in the Pep2–A2-treated A549 and HCT-8 cells (Supplementary Fig. 6g). To further confirm whether it is the p62/TRB3 interaction responsible for tumour-promoting effects of TRB3, we constructed a p62mut expression vector that is p62-siRNA-resistant. This mutant lacks the C-terminal LIR and UBA domains and cannot interact with TRB3, but retains the self-oligomerization action. We found that the Pep2–A2 peptide showed no inhibiting effects on the proliferation and invasion of HepG2 cells with endogenous p62 depletion and isotopic expression of p62mut (Fig. 8e,f). Moreover, Pep2–A2mut peptide could not suppress tumour growth and invasion (Fig. 8g,h). These data demonstrate that the p62/TRB3 interaction is responsible for the tumour-promoting effects of TRB3.